Tenascin-X in amniotic fluid and reproductive tissues of pregnancies complicated by infection and preterm prelabor rupture of membranes†

Abstract

Preterm prelabor rupture of membranes (PPROM), which can precede or follow intra-amniotic infection/inflammation (IAI), is a poorly understood pregnancy complication. Tenascin-X (TNX) is a connective tissue extracellular matrix protein that regulates fibrillogenesis of collagens I, III, and V. Our goal was to investigate the presence and level of soluble TNX (sTNX) in amniotic fluid (AF) and TNX expression in reproductive tissues of pregnancies complicated by PPROM and IAI. We prospectively recruited 334 women pregnant with singletons who had a clinically indicated amniocentesis for genetic karyotyping, lung maturity testing, or rule-out IAI in the presence or absence of PPROM. We quantified TNX expression in fetal membranes, myometrium, cervix, and placenta using immunological methods and qRT-PCR. In pregnancies with normal outcomes, AF sTNX levels were GA-regulated with lower levels toward term. IAI significantly upregulated AF sTNX levels independent of membrane status. AF sTNX levels inversely correlated with fetal membranes tenascin XB (TNXB) mRNA level, which was significantly downregulated by IAI. Western blotting identified characteristic ∼75 and ∼140 kDa sTNX forms in both AF and fetal membranes. Fetal membranes, placenta, and cervix constitutively express TNX with the highest abundance in the amnion. Amnion TNX richness is significantly lost in the setting of IAI. Our results suggest that fetal membranes may be a source of AF sTNX whereby protein and mRNA expression seem to be significantly impacted by inflammation independent of fetal membrane status. A more thorough understanding of TNX changes may be valuable for understanding spontaneous PPROM and to potentially develop therapeutic targets.

Keywords: collagen; fetal membranes; infection; labor; pregnancy.

Figure 1.

(A) Amniotic fluid (AF) mean soluble tenascin-X (sTNX) levels and standard error mean detected in women who had a normal pregnancy outcome (second trimester genetic amniocentesis n = 34, second trimester rule-out infection that delivered at term n = 43, and third trimester lung maturity women n = 31. (B) AF mean sTNX levels and standard error mean detected in women who had an amniocentesis to rule-out intra-amniotic fluid infection/inflammation (IAI) in the setting of intact or preterm prelabor rupture of membranes (PPROM). Samples included in this analysis were from women who delivered preterm. Within each graph, * denotes statistical significance at P < 0.05.

Figure 2.

(A) Representative western blots demonstrating immunoreactive proteoforms of soluble tenascin-X (sTNX) in human serum (lanes 1 and 2) of two women who delivered preterm in the setting of intra-amniotic fluid infection (IAI) and histologic chorioamnionitis. As shown, serum displays TNX immunoreactivity at ∼75 and ∼140 kDa (marked by arrowheads). Negative controls are presented on lanes 3 and 4 where primary antibody was substituted with nonimmune serum. (B) Fetal membranes TNX forms were identified at ∼75 and ∼140 kDa, in a similar fashion to serum and AF (lanes 5–8). (C) Representative western blots of amniotic fluid retrieved from women without (No) or with (Yes) IAI in the setting of either intact or preterm prelabor rupture of membranes (PPROM) (lanes 9–16). In the absence of IAI, the characteristic TNX bands at ∼75 and ∼140 kDa were identified. In several AF samples of women with IAI, the characteristic ∼140 kDa sTNX band was absent independent of membrane status (lanes 13 and 16). In these cases, sTNX species of lower molecular weights were identified. (D) Deglycosylation (DG) of two AF samples (lanes 17 and 19) showed that the sTNX proteoforms were glycosylated, given the shift to a lower molecular weight (lanes 18 and 20).

Figure 3.

(A) Mean tenascin XB (TNXB) and standard error mean mRNA levels in the fetal membranes of women with (Yes) and without (No) intra-amniotic fluid infection/inflammation (IAI) in the setting of either intact or preterm prelabor rupture of membranes (PPROM). (B) Relationship between amniotic fluid (AF) soluble tenascin-X (TNX) levels and TNXB mRNA levels in women with IAI. Thick black line, linear regression line; thick gray lines, 95% confidence intervals; dotted black lines, 95% prediction intervals. (C) Mean TNXB and standard error mean mRNA levels in reproductive tissues (fetal membranes, myometrium, cervix, and placenta). In panel C, the thick line represents the median and graph bars display the interquartile range. Within each graph, * denotes statistical significance at P < 0.05.

Figure 4.

Representative histological images of tenascin-X tissue immunostaining in the human fetal membranes (A), myometrium (B), cervix (C), and placenta (D).

Figure 5.

(A, B) Tissue immunostaining of tenascin-X (TNX) in human fetal membranes of women without (No) or with (Yes) histologic chorioamnionitis (HCA). (C, D) The amnio-epithelium (ae, black arrow) and choriodecidua (cd) of women with No HCA display intense staining for TNX, in strong contrast to women with Yes HCA, who exhibited consistently minimal ae staining. At the interface between ae and cd (see insert), the area surrounding the infiltrating inflammatory cells (green fluorescent arrow heads) is clear of TNX. (E) Mean TNX and standard error mean analysis of the intensity of TNX in the ae of women with and without intra-amniotic fluid infection/inflammation (IAI). (F-H) Double immunostaining of fetal membranes for TNX (green fluorescence) and CD45 (red fluorescence) from a representative case with IAI demonstrating presence of tissue infiltrating leukocytes. Nuclei were counterstained with DAPI. The CD45 + inflammatory cells did not express TNX but disturbed the continuity of tissue TNX immunoreactivity along the fetal membranes. Intracellular TNX staining was observed in ae cells (white arrow). (I) Negative control with omitted primary antibodies. * denotes statistical significance at P < 0.05.